Critical evaluation of epidemiological, virological, and immunological data accumulated during the last decade leads to the inevitable conclusion that HIV-1 infection must be considered primarily as a mucosal disease. The absolute majority of HIV-1 infections are encountered by the mucosal route during vaginal and anal sexual encounters, with women infected at a higher frequency than males. A number of potential mechanisms, addressed experimentally in this proposal, may be involved in the transmission of free and cell associated HIV across mucosal membranes. Penetrating HIV-1 promptly infects subepithelial target cells (mostly CD4+ T cells), resulting in a remarkably extensive depletion of this cell population in mucosal tissues, particularly in the gut and other mucosal organs and tissues including the female genital tract. It is speculated that as a consequence of mucosal T cell depletion and the resulting breakdown of immunoregulatory mechanisms, mucosal defenses are severely impaired and environmental antigens, mainly of bacterial origin, are taken up at much higher rates and activate target cells residing in the systemic immune compartment. Furthermore, numerous studies performed in humans strongly suggest that there is a significant association between the use of progesterone-based humoral contraceptives and a markedly increased risk of HIV-1 infection. The submitted proposal represents an integrated approach focused on a unique compartment of the mucosal immune system - the female genital tract - and HIV-1 infection. Based on the individual components of this application, the overall specific aims of the entire proposal will address: 1) the immunobiology of HIV-1 entry and infection in the female genital tract with respect to the identification of cells and their receptors involved in HIV-1 entry and susceptibility to HIV-1 infection, and the role of antibodies in the prevention of HIV-1 infection;2) marked alterations of humoral responses in the female genital tract with respect to the unexpected paucity of HIV-1-specific IgA responses in infected women, and HIV-1-induced changes in T and B cells with respect to the expression of mucosal and systemic lymphocyte homing receptors;and 3) the impact of progesterone-based contraceptives on mucosal immunity in HIV-1- infected women. The success of these studies is dependent on accessibility to suitable cohorts of women, as specified and described in the Core B section of this proposal. RELEVANCE: The understanding of the biological and immunological consequences of HIV infection on the female genital tract and the effects of hormonal contraception on HIV infection and disease progression is limited. Elucidation of the mechanisms involved in viral entry, immunological, and hormonal interactions in HIV infected and uninfected women will provide critical insight, with implications in the design of strategies for the prevention of new infections and the reduction of HIV-associated morbidity and mortality in women. PROJECT 1: Immunobiology of HIV-1 Entry and Infection in the Female Genital Tract (Project Leader: Smith, P) PROJECT 1 DESCRIPTION (provided by applicant): The immunobiology of HIV-1 infection in the female genital tract involves three major events: (a) Entry through the mucosal epithelium;(b) Infection and subsequent replication in subepithelial mononuclear cells;and (c) Delivery to lymph nodes to initiate systemic infection. To dissect these events, studies of macaque and human genital tissues have yielded variable results, likely because multiple cells may be involved in the events. Moreover, microbicidal agents that block SIV infection in macaques fail to block, or even enhance, infection in women, underscoring the urgent need for an effective mucosal vaccine. Importantly, because women may also acquire HIV-1 through rectal exposure, an effective vaccine for all at-risk women needs to block HIV-1 entry and infection in both genital and rectal mucosa. Using a mucosal explant system and purified mucosal cells, this proposal will elucidate the immunobiology of HIV-1 entry and infection and characterize the effect of anti-HIV-1 antibodies and progesterone-based hormonal agents on these events in genital and rectal mucosa. The proposal is driven by four Hypotheses: (1) HIV-1 crosses the monostratified endocervical and rectal epithelium by epithelial cell transcytosis but crosses pleuristratified ectocervical and vaginal epithelium via DCs;(2) Female genital mucosa selects the R5, genotypically restricted viruses that characterize acute HIV-1 infection;(3) In female genital mucosa, macrophages, lymphocytes and DCs are permissive to HIV-1 infection, but in rectal mucosa only lymphocytes and DCs are permissive;and (4) HIV- 1 entry and infection in genital and rectal mucosae are inhibited by IgG (and possibly IgA) anti-HIV-1 antibodies, and receptor analogs and ligands. These hypotheses will be tested with four Specific Aims: (1) Determine the cell(s), attachment molecule(s) and receptor(s) that cell-free and cell-associated R5 and X4 HIV-1 utilizes to enter the endocervical, ectocervical, vaginal and rectal epithelium. (2) Determine whether genital mucosa selects the R5 viruses that characterize acute HIV-1 infection. (3) Determine whether HIV-1 in female genital mucosa infects lymphocytes, macrophages and DCs but in rectal mucosa infects only lymphocytes and DCs. (4) Determine whether anti-HIV-1 (gp41 GalCer-binding domain, gp41, gp120) antibodies and CCR5 anti-virals block cell-free and cell-associated R5 and X4 HIV-1 entry and target cell infection in female genital and rectal mucosa. RELEVANCE: This proposal will use primary mucosal tissues and purified mucosal cells to define the key events in HIV-1 entry and infection in human female genital and rectal tissue in the presence and absence of progesterone based hormonal agents. The ability of antibodies and anti-receptor agents to block these events will be characterized, thereby providing critical information for the development of a mucosal vaccine to prevent genital and rectal HIV-1 infection in women.